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SipB-SipC complex is essential for translocon formation.

ABSTRACT: The delivery of effector proteins by Salmonella across the host cell membrane requires a subset of effectors secreted by the type III secretion system (TTSS) known as translocators. SipC and SipB are translocator proteins that are inserted into host membranes and presumably form a channel that translocates type III effectors into the host cell. The molecular events of how these translocators insert into the host cell membrane remain unknown. We have previously shown that the SipC C-terminal amino acid region (321-409) is required for the translocation of effectors into host cells. In this study, we demonstrate that the ability to form SipC-SipB complex is essential for their insertion into the host membrane. The SipB-interacting domain of SipC is near its C-terminal amino acid region (340-409). In the absence of SipB, SipC was not detected in the membrane fraction. Furthermore, SipC mutants that no longer interact with SipB are defective in inserting into the host cell membrane. We propose a mechanism whereby SipC binds SipB through its C-terminal region to facilitate membrane-insertion and subsequent translocon formation in the host cell membrane.

SUBMITTER: Myeni SK

PROVIDER: S-EPMC3609803 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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SipB-SipC complex is essential for translocon formation.

Myeni Sebenzile K SK Wang Lu L Zhou Daoguo D

PloS one 20130327 3

The delivery of effector proteins by Salmonella across the host cell membrane requires a subset of effectors secreted by the type III secretion system (TTSS) known as translocators. SipC and SipB are translocator proteins that are inserted into host membranes and presumably form a channel that translocates type III effectors into the host cell. The molecular events of how these translocators insert into the host cell membrane remain unknown. We have previously shown that the SipC C-terminal amin ...[more]

PMID: 23544147

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Project description:The type III secretion system (T3SS) is a syringe-like virulence factor that delivers bacterial proteins directly into the cytoplasm of host cells. An essential component of the system is the translocon, which creates a pore in the host cell membrane through which proteins are injected. In Pseudomonas aeruginosa, the translocation pore is formed by proteins PopB and PopD and attaches to the T3SS needle via the needle tip protein PcrV. The structure and stoichiometry of the multimeric pore are unknown. We took a genetic approach to map contact points within the system by taking advantage of the fact that the translocator proteins of P. aeruginosa and the related Aeromonas hydrophila T3SS are incompatible and cannot be freely exchanged. We created chimeric versions of P. aeruginosa PopB and A. hydrophila AopB to intentionally disrupt and restore protein-protein interactions. We identified a chimeric B-translocator that specifically disrupts an interaction with the needle tip protein. This disruption did not affect membrane insertion of the B-translocator but did prevent formation of the translocation pore, arguing that the needle tip protein drives the formation of the translocation pore. IMPORTANCE Type III secretion systems are integral to the pathogenesis of many Gram-negative bacterial pathogens. A hallmark of these secretion systems is that they deliver effector proteins vectorially into the targeted host cell via a translocation pore. The translocon is crucial for T3SS function, but it has proven difficult to study biochemically and structurally. Here, we used a genetic approach to identify protein-protein contacts among translocator proteins that are important for function. This genetic approach allowed us to specifically break a contact between the translocator PopB and the T3SS needle tip protein PcrV. Breaking this contact allowed us to determine, for the first time, that the needle tip actively participates in the assembly of the translocation pore by the membrane-bound pore-forming translocator proteins. Our study therefore both expands our knowledge of the network of functionally important interactions among translocator proteins and illuminates a new step in the assembly of this critical host cell interface.

Project description:Spores are the major infectious particle of the Gram-positive nosocomial pathogen Clostridioides difficile (formerly Clostridium difficile), but the molecular details of how this organism forms these metabolically dormant cells remain poorly characterized. The composition of the spore coat in C. difficile differs markedly from that defined in the well-studied organism Bacillus subtilis, with only 25% of the ?70 spore coat proteins being conserved between the two organisms and with only 2 of 9 coat assembly (morphogenetic) proteins defined in B. subtilis having homologs in C. difficile We previously identified SipL as a clostridium-specific coat protein essential for functional spore formation. Heterologous expression analyses in Escherichia coli revealed that SipL directly interacts with C. difficile SpoIVA, a coat-morphogenetic protein conserved in all spore-forming organisms, through SipL's C-terminal LysM domain. In this study, we show that SpoIVA-SipL binding is essential for C. difficile spore formation and identify specific residues within the LysM domain that stabilize this interaction. Fluorescence microscopy analyses indicate that binding of SipL's LysM domain to SpoIVA is required for SipL to localize to the forespore while SpoIVA requires SipL to promote encasement of SpoIVA around the forespore. Since we also show that clostridial LysM domains are functionally interchangeable at least in C. difficile, the basic mechanism for SipL-dependent assembly of clostridial spore coats may be conserved.IMPORTANCE The metabolically dormant spore form of the major nosocomial pathogen Clostridioides difficile is its major infectious particle. However, the mechanisms controlling the formation of this resistant cell type are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore-morphogenetic proteins in C. difficile: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL's C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and, thus, functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious C. difficile spores.

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SipB-SipC complex is essential for translocon formation.

Publications

SipB-SipC complex is essential for translocon formation.

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